Systems and methods for generating droplets and performing digital analyses

ABSTRACT

This disclosure provides for devices, methods, and systems for generating a plurality of droplets within a collecting container at an extremely high rate (e.g., of at least 1 million droplets per minute, etc.), each of the plurality of droplets comprising an aqueous mixture for a digital analysis, wherein upon generation, the plurality of droplets is stabilized in position within a region of the collecting container. The inventions enable partitioning of samples for digital analyses at unprecedented rates, where readout of signals from targets within such partitions can still be achieved in accordance with various assays.

CROSS-REFERENCE

This application is a continuation application of PCT Patent Applicationnumber PCT/US2022/018994, filed Mar. 4, 2022, which claims the benefitof U.S. Provisional Application No. 63/157,292, filed Mar. 5, 2021, eachof which is incorporated in its entirety herein by this reference.

TECHNICAL FIELD

This disclosure relates generally to fields related to sample processingand digital analyses, and more specifically to a new and useful systemsand methods for generation of and digital analysis of partitions in suchfields.

BACKGROUND OF THE INVENTION

In biotechnology and other applications, partitioning technologies playa significant role in achieving the ability to conduct microscale andnanoscale analyses (e.g., of single cells, of single molecules, of otheranalytes, etc.). Dispersing samples across partitions in a consistentand reliable manner has utility in relation to various assays in celland molecular biology, with respect to digital analyses (e.g., digitalpolymerase chain reaction) and other bioassays. Isolated and independentreaction environments provided in a partitioned-format can furthergreatly reduce the sample and process fluid volume required, reducingcosts associated with sample processing. Results returned from suchassays can be used for clinical and non-clinical characterizations ofvarious conditions.

SUMMARY OF THE INVENTION

Currently, methods and systems for distributing samples acrosspartitions in a rapid and consistent manner, in a manner where partitioncontents are stably isolated without leakage or merging of contents ofadjacent partitions, and in a manner that enables readout from signalsfrom the partitions, are severely limited. Commercially availableplatforms conduct partitioning by using microfluidic devices involvingcomplex setups. However, such platforms may be costly, partition samplesat a slow rate, are labor-intensive, or can cause sample contamination.Thus, there is a need in the field of sample processing to create newand useful systems and methods for generation of and digital analysis ofpartitions.

Accordingly, this disclosure describes embodiments, variations, andexamples of systems, methods and compositions for rapid partitioning ofsamples for digital analyses.

An aspect of the disclosure provides embodiments, variations, andexamples of a device for rapidly generating partitions (e.g., dropletsfrom a sample fluid, droplets of an emulsion), wherein, the deviceincludes: a first substrate defining a reservoir comprising a reservoirinlet and a reservoir outlet; a membrane coupled to the reservoir outletand comprising a distribution of holes; and a supporting body comprisingan opening configured to retain a collecting container in alignment withthe reservoir outlet. During operation, the first substrate can becoupled with the supporting body and enclose the collecting container,with the reservoir outlet aligned with and/or seated within thecollecting container. During operation, the reservoir can contain asample fluid, where application of a force to the device or sample fluidgenerates a plurality of droplets within the collecting container at anextremely high rate (e.g., of at least 200,000 droplets/minute, of atleast 300,000 droplets/minute, of at least 400, droplets/minute, of atleast 500,000 droplets/minute, of at least 600,000 droplets/minute, ofat least 700,000 droplets/minute, of at least 800,000 droplets/minute,of at least 900,000 droplets/minute, of at least 1 milliondroplets/minute, of at least 2 million droplets/minute, of at least 3million droplets/minute, etc.), where the droplets are stabilized inposition (e.g., in a close-packed format, in equilibrium stationarypositions) within the collecting container. Notably, the droplets arestable across a wide range of temperatures (e.g., 1° C. through 95° C.,greater than 95° C., less than 1° C.) relevant to various digitalanalyses and other bioassays, where the droplets remain consistent inmorphology and remain unmerged with adjacent droplets.

In embodiments, variations, and examples, the membrane includes adistribution of holes having low density (e.g., significantly lower thanthat typical for filtration applications involving porous membranes).

In embodiments, variations, and examples, the device can include a setof reservoirs (e.g., at the first substrate), a set of membranes atoutlets of the set of reservoirs, and a supporting body for a set ofcollecting containers, in order to provide parallel processing ofmultiple samples and/or combination of sample processing materialsduring the partitioning process.

In embodiments, variations, and examples, the device can include aspacer configured to separate the membrane(s) further from base surfacesor liquid interfaces within the collecting container(s), therebyenabling operation modes in which droplets emerging from the membranepass through air or another initial fluid phase prior to arriving atrespective equilibrium positions.

An aspect of the disclosure provides embodiments, variations, andexamples of a method for rapidly generating partitions (e.g., dropletsfrom a sample fluid, droplets of an emulsion) within a collectingcontainer at an extremely high rate, each of the plurality of dropletsincluding an aqueous mixture for a digital analysis (e.g., of nucleicacid material, of protein material, of amino acid material, of otheranalytes described), wherein upon generation, the plurality of dropletsis stabilized in position (e.g., in a close-packed format, atequilibrium stationary positions, etc.) within a continuous phase (e.g.,as an emulsion having a bulk morphology defined by the collectingcontainer). In aspects, partition generation can be executed by drivingthe sample fluid through a distribution of holes of a membrane, wherethe applied force can be one or more of centrifugal, associated withapplied pressure, magnetic, or otherwise physically applied.

In embodiments, variations, and examples, droplets generated can form anemulsion, with individual droplets isolated and stabilized in positionwithin a collecting container. The emulsion can be a viscous fluid, ashear-thickening fluid, a gel (e.g., a gel having individual discretedroplets), or another fluid having a surface. When droplet generation isperformed by way of centrifugation according to variations of themethod(s) described, driving the sample fluid through the membrane caninclude spinning the sample fluid, the membrane, and the collectingcontainer in a first direction of rotation, and reversing the directionof rotation, thereby adjusting a surface profile of an emulsioncomprising the plurality of droplets within the collecting container. Inrelation to digital analyses involving optical interrogation of sectionsof the emulsion, adjusting the surface profile (e.g., by producing amore even/level/planar surface during centrifugation) can improvereadout of signals proximal to all surfaces of the emulsion.

When droplet generation is performed by way of centrifugation accordingto variations of the method(s) described, driving the sample fluidthrough the membrane can include. spinning the sample fluid, themembrane, and the collecting container within a centrifuge at a firstrotational velocity and at a second rotational velocity less than thefirst rotational velocity, thereby adjusting a surface profile of anemulsion comprising the plurality of droplets within the collectingcontainer. In relation to digital analyses involving opticalinterrogation of sections of the emulsion, adjusting the surface profile(e.g., by producing a more even/level/planar surface duringcentrifugation) can improve readout of signals proximal to all surfacesof the emulsion.

In relation to a single-tube workflow in which the collecting containerremains closed (e.g., the collecting container has no outlet, there isno flow out of the collecting container, to avoid sample contamination),method(s) can further include transmitting heat to and from theplurality of droplets within the closed collecting container accordingto an assay protocol. In relation to generation of emulsions havingsuitable clarity (e.g., with or without refractive index matching),method(s) can further include transmission of signals from individualdroplets from within the closed collecting container, for readout (e.g.,by an optical detection platform, by another suitable detectionplatform).

Where method(s) include transmitting heat to and from the plurality ofdroplets, within the closed container, the droplets are stable across awide range of temperatures (e.g., 1° C. through 95° C., greater than 95°C., less than 1° C.) relevant to various digital analyses and otherbioassays, where the droplets remain consistent in morphology and remainunmerged with adjacent droplets.

Examples of partition generation methods can include generating anextremely high number of droplets (e.g., greater than 2 milliondroplets, greater than 3 million droplets, greater than 4 milliondroplets, greater than 5 million droplets, greater than 6 milliondroplets, greater than 7 million droplets, greater than 8 milliondroplets, greater than 9 million droplets, greater than 10 milliondroplets, greater than 15 million droplets, greater than 20 milliondroplets, greater than 25 million droplets, greater than 30 milliondroplets, greater than 40 million droplets, greater than 50 milliondroplets, greater than 100 million droplets, etc.) within a collectingcontainer having a volumetric capacity (e.g., less than 50 microliters,from 50 through 100 microliters and greater, etc.), where droplets havea characteristic dimension (e.g., from 1-50 micrometers, from 10-30micrometers, etc.) that is relevant for digital analyses, single cellcapture, target detection, individual molecule partitioning, or otherapplications.

The disclosure provides for systems, devices, and methods that enabledigital analyses across a wide dynamic range that is 10-100 timesgreater than that of existing technologies, depending upon applicationof use. In examples related to nucleic acid counting, the disclosureprovides for systems, devices, and methods that can have a dynamic rangefrom 1 through 100 million, due to the extremely high number of uniformpartitions generated from which signals can be read, and due to theability to partition with low occupancy (e.g., less than 20% occupancy,less than 10% occupancy, less than 9% occupancy, less than 8% occupancy,less than 7% occupancy, less than 6% occupancy, less than 5% occupancy,etc.) of partitions by targets.

In specific applications, partitioning devices and methods described canperform: detection and counting of nucleic acid molecules viaamplification of individual nucleic acid molecule captured within adroplet followed by detection of optically detectable signals (e.g.,amplification by polymerase chain reaction (PCR) methods, by isothermalmethods such as loop-mediated isothermal amplification (LAMP), byrecombinase polymerase amplification (RPA), by helicase dependentamplification (HDA), by strand displacement amplification (SDA), bynicking enzyme amplification (NEAR), by transcription mediatedamplification (TMA), by RNaseH mediated amplification, by whole genomeamplification (WGA) using phi29, by rolling circle amplification, etc.)on purified DNA, cDNA, RNA, oligonucleotide taggedantibodies/proteins/small molecules, or directly from lysate (e.g.,blood lysate); fluorescent in situ hybridization (FISH) withfluorescently tagged nucleic acids (e.g., PNA, LNA, DNA, RNA, etc.) oran indirect in situ hybridization approach using DIG or biotin, wherethe signal is later amplified by conjugation of an antibody to alkalinephosphatase or a peroxidase to produce a change in color detected by oneor more substrates (e.g., nitroblue tetrazolium (NBT),5-bromo-4-chloro-3-indolyl-phosphate (BCIP), HNPP, etc.); an in vitrotranscription or translation assay whereby a colorimetric or fluorescentreporter is used for detection; droplet PCR applied to samples derivedfrom single cells (e.g., prokaryotes, eukaryotes), organelles, viralparticles, and exosomes; enumeration of protein or peptide molecules(e.g., by proximity ligation assays, etc.); sequencing applications(e.g., single molecule sequencing applications); monitoring or detectionof products (e.g., proteins, chemicals) released from single cells(e.g., interleukin released from immune cells); monitoring cell survivaland/or division for single cells; monitoring or detection of enzymaticreactions involving single cells; antibiotic resistance screening forsingle bacteria; enumeration of pathogens in a sample (e.g., in relationto infections, sepsis, in relation to environmental and food samples,etc.); enumeration of heterogeneous cell populations in a sample;enumeration of individual cells or viral particles (e.g., byencapsulating cells in droplets with species-specific antibodies coupledwith enzymes that react with substrate components in the droplet toproduce signals, etc.); monitoring of viral infections of a single hostcell; liquid biopsies and companion diagnostics; prenatal diagnosis ofgenetic disorders (e.g., aneuploidy, genetically inherited diseases)such as with cell-free nucleic acids, fetal cells, or samples containingmixtures of fetal and maternal cells; detection of cancer forms fromvarious biological samples (e.g., detection of cancer from cell-freenucleic acids, tissue biopsies, biological fluids, feces); detectionand/or monitoring of minimal residual diseases; monitoring responses totherapies; detection or prediction of rejection events of transplantedorgans; other diagnostics associated with other health conditions; othercharacterizations of statuses of other organisms; and other suitableapplications.

In specific applications, the systems and methods for partitioning in asingle tube workflow can perform emulsion digital PCR-associatedprocesses.

In embodiments, the target material analyzed according to digitalanalysis and/or other bioassay techniques can include one or more of:nucleic acid material (e.g., DNA, RNA, miRNA, etc.), protein material,amino acid material, other small molecules, other single analytes, othermulti-analytes, and/or other suitable target material of a sample. Inembodiments, the sample can include or otherwise be derived from wholetissue structures, tissue portions (e.g., histological tissue slices,formalin-fixed paraffin-embedded (FFPE) tissue, frozen tissue, biopsiedtissues, fresh frozen plasma, seeded natural scaffolds, seeded syntheticscaffolds, etc.), organs, whole organisms, organoids, cell suspensions(e.g., frozen cell suspensions that are separated prior to processingwith the system, cell suspensions retained in a medium/hydrogel medium,etc.), nuclei suspension, single cells, organelles, sub-organellestructures, intra-organelle components, viruses, microorganisms, andother samples.

An additional aspect of the present disclosure provides for a methodcomprising: generating a plurality of droplets within a collectingcontainer at a rate of at least 1 million droplets per minute, each ofthe plurality of droplets comprising an aqueous mixture for a digitalanalysis of nucleic acid material.

In some embodiments, upon generation, the plurality of droplets isstabilized in position in a close-packed format within a continuousphase, within a region of the collecting container. In some embodiments,generating the plurality of droplets comprises driving a sample fluidthrough a membrane comprising a distribution of holes, the membranecoupled to a reservoir outlet of a reservoir for the sample fluid, andthe reservoir aligned with the collecting container. In someembodiments, the distribution of holes has a density less than 5000holes per cm² and a hole-to-hole spacing greater than 30 micrometers. Insome embodiments, each hole in the distribution of holes has a diameterfrom 1 through 3 micrometers. In some embodiments, driving the samplefluid through the membrane comprises spinning the sample fluid, themembrane, and the collecting container within a centrifuge in a firstdirection of rotation, and reversing the direction of rotation, therebyadjusting an equilibrium surface profile of an emulsion comprising theplurality of droplets within the collecting container. In someembodiments, driving the sample fluid through the membrane comprisesspinning the sample fluid, the membrane, and the collecting containerwithin a centrifuge at a first rotational velocity and at a secondrotational velocity less than the first rotational velocity, therebyadjusting an equilibrium surface profile of an emulsion comprising theplurality of droplets within the collecting container. In someembodiments, the collecting container has a volumetric capacity from 10through 300 microliters, and wherein each of the plurality of dropletshas a characteristic diameter from 10 through 30 micrometers. In someembodiments, the method further comprises transmitting heat to and fromthe plurality of droplets, within the collecting container, during aheat transmission operation, wherein the temperature varies within atemperature range from 4° C. to 95° C. during the heat transmissionoperation, and wherein individual droplets of the plurality of dropletsremain unmerged with adjacent droplets in a close-packed format duringthe heat transmission operation. In some embodiments, generating theplurality of droplets comprises generating greater than 25 milliondroplets within the collecting container. In some embodiments,generating the plurality of droplets comprises transmitting twodimensional arrays of droplets toward a closed end of the collectingcontainer, thereby stabilizing the plurality of droplets in a threedimensional close-packed format toward the closed end of the collectingcontainer.

An additional aspect of the present disclosure provides for a system forgenerating droplets, the system comprising: a first substrate defining areservoir comprising a reservoir inlet and a reservoir outlet; amembrane coupled to the reservoir outlet and comprising a distributionof holes; and a second substrate comprising an opening configured toretain a collecting container in alignment with the reservoir outlet,wherein the system comprises: a first operation mode wherein the firstsubstrate is coupled with the second substrate and encloses thecollecting container, with the reservoir outlet seated within thecollecting container, a second operation mode wherein the reservoircontains a sample fluid comprising an aqueous mixture for a digitalanalysis of nucleic acid material, and a third operation mode whereinthe membrane generates a plurality of droplets within the collectingcontainer at a rate of at least 1 million droplets per minute inresponse to a force applied to the sample fluid, and a fourth operationmode wherein the plurality of droplets is stabilized in position in aclose-packed format within a region of the collecting container.

In some embodiments, the membrane is bonded to the reservoir outlet at aperimeter of the reservoir outlet. In some embodiments, the distributionof holes has a density less than 10,000 holes per cm² and a hole-to-holespacing greater than 10 micrometers. In some embodiments, the firstsubstrate comprises a set of reservoirs comprising the reservoir; andthe system further comprises a set of membranes comprising the membrane,the set of membranes paired with and bonded to outlets of the set ofreservoirs; and the second substrate comprises a set of openingscomprising the opening, wherein the set of openings is configured toretain a set of collecting containers in alignment with the set ofreservoirs. In some embodiments, a reservoir number of the set ofreservoirs is different from a collecting container number of the set ofcollecting containers, wherein the first substrate comprises two or morefluidic pathways from the set of reservoirs to the set of membranes. Insome embodiments, the second substrate is configured as a spacerseparating the reservoir outlet from a base surface of the collectingcontainer.

An additional aspect of the present disclosure provides for a methodcomprising: generating a plurality of droplets within a collectingcontainer, each of the plurality of droplets comprising an aqueousmixture for a digital analysis of nucleic acid material, whereingenerating the plurality of droplets comprises driving the aqueousmixture through a distribution of holes of a track-etched membrane tostabilized positions, toward a closed end of the collecting container,and wherein the plurality of droplets is characterized by less than 13%coefficient of variation for polydispersity.

In some embodiments, generating the plurality of droplets comprisesgenerating the plurality of droplets at a rate of at least 600,000droplets per minute. In some embodiments, the plurality of droplets ischaracterized by less than 10% occupancy of droplets by said nucleicacid material. Another aspect of the present disclosure provides anon-transitory computer readable medium comprising machine executablecode that, upon execution by one or more computer processors, implementsany of the methods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. The present disclosure iscapable of other and different embodiments, and its several details arecapable of modifications in various obvious respects, all withoutdeparting from the disclosure. Accordingly, the drawings and descriptionare to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entiretiesfor all purposes and to the same extent as if each individualpublication, patent, or patent application was specifically andindividually indicated to be incorporated by reference.

Furthermore, where a range of values is provided, it is understood thateach intervening value, between the upper and lower limit of that rangeand any other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an embodiment of a system for generating droplets.

FIG. 2 depicts a first example of a system for generating droplets.

FIG. 3 depicts a variation of a fastener configuration in a system forgenerating droplets.

FIG. 4 depicts a variation of a fastener configuration in a system forgenerating droplets.

FIG. 5 depicts an example of a method for assembling components of asystem for generating droplets.

FIG. 6 depicts a variation of a configuration of a system for generatingdroplets.

FIG. 7A depicts a second example of a system for generating droplets.

FIG. 7B depicts a third example of a system for generating droplets.

FIG. 7C depicts examples of collecting containers of a system forgenerating droplets.

FIG. 8 depicts a flow chart of an embodiment of a method for generatingdroplets.

FIGS. 9A-9C depict examples of methods for producing desired surfacecharacteristics of emulsions generated according to methods described.

FIG. 10 depicts a flow chart of an embodiment of a method for generatingdroplets.

FIG. 11 illustrates a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

DETAILED DESCRIPTION OF THE INVENTION(S)

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions can occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein can beemployed.

1. GENERAL OVERVIEW

The present disclosure covers systems, devices, methods performed bysuch systems and devices, and methods of manufacturing and assemblingsuch devices. Generally, embodiments of the device include assemblies ofreservoirs, functionalized membranes, and supporting bodies forcollecting containers, where the assemblies rapidly produce droplets ofan emulsion for digital analyses or other applications of use. Dropletsproduced by such devices are stabilized in a three dimensional formatwithin closed collecting containers, thereby providing a “single-tube”workflow that eliminates risk of sample cross-contamination from initialreception of a sample, to distributing of the sample across an extremelyhigh number of partitions, to performance of reactions within individualpartitions, to detecting signals generated by contents of individualpartitions from with the closed collecting containers.

The systems, methods, and devices disclosed herein can provide severaladditional benefits over other systems and methods, and such systems,methods, and devices are further implemented into many practicalapplications across various disciplines.

Devices, methods, and systems of the present disclosure may generate aplurality of droplets at an extremely high rate, where the droplets arestabilized in position (e.g., in a close-packed format, in equilibriumstationary positions) within a collecting container. Notably, thedroplets are stable across a wide range of temperatures (e.g., 1° C.through 95° C., greater than 95° C., less than 1° C.) relevant tovarious digital analyses and other bioassays, where the droplets remainconsistent in morphology and remain unmerged with adjacent droplets.Stabilization of the droplets within a continuous phase of an emulsionis further performed in a manner where the emulsion has a high degree ofclarity (e.g., greater than 50% transmissivity of light, greater than60% transmissivity of light, greater than 70% transmissivity of light,greater than 80% transmissivity of light, greater than 90%transmissivity of light, greater than 99% transmissivity of light,etc.), such that signals from cross-sections of the emulsion within thecollecting container can be interrogated (e.g., using a 3D imagingtechnique, using a planar imaging technique, etc.).

The devices, systems, and methods disclosed herein can further generatedroplets in a consistent and controlled manner (e.g., as monodisperse,uniform droplets with a low degree of polydispersity) for applicationsin biotechnology (e.g., with respect to microscale and nanoscale assays)or other fields.

The devices, systems, and methods disclosed herein further provide acost-effective alternative to droplet generation using microfluidicdevices, in a manner that generates droplets in a consistent andreliable manner. In specific examples, the systems and devices mayinclude assemblies with track-etched membranes for producing droplets ina consistent and controlled manner (e.g., as monodisperse/uniformdroplets). In these examples, hole density and/or hole-to-hole spacingof the membranes is significantly lower than that which may used formembrane-based filtration.

The present disclosure also provides disposable devices and methods forgenerating droplets using a single-tube and closed tube workflow fromdroplet generation through reaction performance within individualdroplets, through optical interrogation of droplet contents, therebypreventing sample cross contamination throughout digital analysesprocesses.

Additionally or alternatively, in variations, the devices, methods, andsystems disclosed herein can be adapted for transmitting an extremelyhigh number of droplets, where the droplets are generated at anextremely high rate, to various well formats and tube formats, therebyimproving performance of existing droplet-based systems.

In examples, the systems, methods, and devices of the present disclosurehave applications in digital amplification of nucleic acid molecules(e.g., digital polymerase chain reaction (PCR), digital loop-mediatedisothermal amplification (LAMP), digital multiple displacementamplification (MDA), digital recombinase polymerase amplification (RPA),digital helicase dependent amplification, reverse transcription, invitro transcription and translation, overlap extension amplification,etc.).

In examples, the systems, methods, and devices of the present disclosurehave applications in single cell and organelle capture (e.g., formammalian cells, for bacterial cells, for pathogens, for viralparticles, for organelles, etc.).

In examples, the systems, methods, and devices of the present disclosurehave applications in single, double, or other emulsion generation. Forinstance, the systems, methods, and devices disclosed herein can use oneor more of centrifugation, pressure, or other forces to disperse afluid, as droplets, through one or more layers of fluids (i.e.,‘continuous fluids’) that are immiscible with each other, formingemulsions.

In examples, the systems, methods, and devices of the present disclosurecan have applications in microparticle generation and liposomegeneration for other applications.

Additionally or alternatively, the systems, devices, or methodsdescribed can confer any other suitable benefit.

2. SYSTEMS

As shown in FIG. 1, an embodiment of a system 100 for generation ofdroplets includes: a first substrate 110 defining a set of reservoirs114, each having a reservoir inlet 115 and a reservoir outlet 116; oneor more membranes 120 positioned adjacent to reservoir outlets of theset of reservoirs 114, each of the one or more membranes 120 including adistribution of holes 125; and optionally, a sealing body 130 positionedadjacent to the one or more membranes 120 and including a set ofopenings 135 aligned with the set of reservoirs 114; and optionally, oneor more fasteners (including fastener 140 shown in FIG. 1) configured toretain the first substrate 110, the one or more membranes 120, andoptional the sealing body 130 in position relative to a set ofcollecting containers 150. In variations, the system 100 canadditionally include a second substrate 160, wherein the one or moremembranes 120 and optionally, the sealing body 130, are retained inposition between the first substrate 110 and the second substrate 160 bythe one or more fasteners.

Embodiments of the system 100 function to generate a plurality ofdroplets at an extremely high rate (e.g., of at least 200,000droplets/minute, of at least 300,000 droplets/minute, of at least 400,droplets/minute, of at least 500,000 droplets/minute, of at least600,000 droplets/minute, of at least 700,000 droplets/minute, of atleast 800,000 droplets/minute, of at least 900,000 droplets/minute, ofat least 1 million droplets/minute, of at least 2 milliondroplets/minute, of at least 3 million droplets/minute, etc.), where thedroplets are stabilized in position (e.g., in a close-packed format, inequilibrium stationary positions) within a collecting container. Ratesof droplet generation can be average rates determined in relation toduration of applied force.

Embodiments of the system may further function to reliably generatedroplets in a consistent and controlled manner (e.g., as monodisperseand uniform droplets having little-to-no polydispersity) for variousapplications, such as digital amplification and analysis and otherassays; capture of target material at cellular, subcellular, andmolecular scales; sample analysis benefitting from droplet generation;or other suitable applications. Embodiments of the system 100 alsofunction to generate droplets using devices that are non-microfluidic,disposable or reusable, in a cost-effective manner.

Embodiments of the system 100 can be used to implement one or more stepsof methods described in Section 3 below. However, the system 100 canadditionally or alternatively be configured to perform other suitablemethods.

2.1 System—Supporting Substrates/Housing 2.1.1 SupportingSubstrates—First Substrate

FIG. 1 depicts an embodiment of a system 100, where the system 100includes a first substrate 110 defining a set of reservoirs 114, eachhaving a reservoir inlet 115 and a reservoir outlet 116. The firstsubstrate 110 can function to provide at least a portion of a couplingmechanism for retaining positions of other elements of the system 100relative to one or more collecting containers 150 described in moredetail below. The first substrate 110 also functions to receive andstage fluid to be transmitted through the membrane layer 120 forformation of droplets. The first substrate 110 can be configured todisposable (e.g., composed of inexpensive, recyclable, and/orcompostable materials), such that the system 100 can provide acost-effective alternative for generation of droplets. Additionally oralternatively, the first substrate 110 can be configured to be areusable element (e.g., usable for generation of droplets in multipleruns of the system 100).

In order to provide a robust mechanism of coupling with other systemelements (e.g., supporting bodies for the collecting containers 150, asecond substrate 160 described below, etc.), the first substrate 110 canbe composed of a material having suitable mechanical properties. Invariations, materials of the first substrate 110 can be configured toprovide suitable mechanical properties in relation to stressesattributed to flow through set of reservoirs 114 (e.g., stresses due tocentrifugation, stresses due to pressurization, radial stresses, shearstresses, longitudinal stresses, tensile stresses, compressive stresses,stresses associated with impacts to the system 100 during use; stressesdue to thermal expansion, stresses due to thermal contraction, and otherassociated stresses depending upon applications of use).

Additionally or alternatively, the first substrate 110 can be composedof a material having suitable thermal properties. In variations,materials of the first substrate no can be configured to providesuitable thermal properties in relation to one or more of: thermalconductivity (e.g., in relation to heating or cooling of fluids fromwhich droplets are generated by the system 100) and other thermalproperties depending upon application of use.

Additionally or alternatively, the first substrate 110 can be composedof a material having suitable physical or surface properties. Invariations, materials of the housing 121 and/or other aspects of theinterface 120 can be configured to provide suitable physical or surfaceproperties in relation to one or more of: non-reactiveness with samplematerials; low porosity (e.g., so as to not absorb sample material);high hydrophobicity; and other suitable physical or surface properties.

Additionally or alternatively, the first substrate 110 can be composedof a material having suitable optical properties (e.g., for opticalinterrogation of sample materials, for protecting sample materials fromelectromagnetic radiation, etc.). In variations, the first substrate 110is translucent, transparent, or otherwise has a high degree oftransparency, to support optical interrogation of sample materials orsample processing materials within the set of reservoir(s) 114.Furthermore, translucent or transparent characteristics of the firstsubstrate 110 may enable operation modes in which contents of the set ofreservoirs 114 can be visualized (e.g., during manual filling, duringautomatic filling), such that a level of material within each reservoircan be verified prior to generation of partitions using the system 100.As such, the first substrate 110 can be composed of a material having asuitable level of transparency. Still alternatively, the first substrate110 can be composed of an opaque material.

In variations, the first substrate 110 can be composted of a syntheticmaterial or a natural material. In examples, the first substrate 110 canbe composed of a polymeric material (e.g., a polyetheretherketone, anacetal, an acrylonitrile butadiene styrene, a nylon, a polycarbonate, apolypropylene, a polystyrene, etc.); a metallic material, a ceramicmaterial, a composite material, or another suitable material. The firstsubstrate 110 can be fabricated by machining, printing (e.g., 3Dprinting), molding (e.g., injection molding), or through anothersuitable method.

In variations, the first substrate 110 can have a broad surface at whichaccess (e.g., through openings) into the set of reservoirs 114 isprovided. As shown in FIG. 1, each reservoir can have a reservoir inlet115 and a reservoir outlet 116. The reservoir inlet 115 can be definedas an open surface at the broad surface of the first substrate 110, andthe reservoir outlet can be defined as an open surface at a secondsurface of the first substrate 110. Each reservoir can define a cavityfor receiving fluid from which droplets are generated. In examples thecavity can have a volume from 1 μL through 100 mL; however, variationsof the cavity can have another suitable volume. The cavity can beconstant in cross section (e.g., transverse cross section) from thereservoir inlet 115 to the reservoir outlet 116. Alternatively, thecavity may not be constant in cross section from the reservoir inlet 115to the reservoir outlet 116. For instance, a portion of the cavity nearthe reservoir inlet 115 can include morphology (e.g., flared morphology)configured to complement a fluid delivery device (e.g., a pipette tip),thereby facilitating fluid transfer to the system 100 for dropletgeneration. Additionally or alternatively, the cavity can define anon-straight or non-linear path from the reservoir inlet 115 to thereservoir outlet 116. Furthermore, in variations, a reservoir of the setof reservoirs 114 can have a reservoir inlet 115 at any other suitablesurface of the first substrate 110, a reservoir outlet 116 at any othersuitable outlet of the first substrate 110, or define another suitablefluid path from the reservoir inlet 115 to the reservoir outlet 116.

One or more filters can further be positioned within the set ofreservoirs 114 and/or otherwise positioned upstream of the one or moremembranes 120 described in further detail below, where the filter(s) canfunction to remove undesired material and prevent undesired materialfrom entering droplets or disrupting droplet formation. In examples, thefilter(s) can be structured to allow passage of targets (e.g., nucleicacids, proteins, cells, viral material, chemicals, analytes, etc.) fromthe sample(s) for distribution across the plurality of droplets, wheresuch filter(s) can accordingly have suitable sizes, porosity,hydrophobicity, surface charge, and/or other characteristics.

The example of the first substrate 110 a shown in FIG. 2 includes fourreservoirs corresponding to four collecting containers (e.g., of acomplementary apparatus) for generated droplets; however, the firstsubstrate 110 can alternatively define another suitable number ofreservoirs (e.g., from one to 10000 reservoirs). Furthermore, the numberof reservoirs may not correspond to the number of collecting containersin a one-to-one manner. For instance, the first substrate 110 can definea number of reservoir inlets fewer than that of the number of collectingcontainers, where the reservoirs can be configured to branch along theirrespective lengths and terminate at outlets corresponding to thecollecting containers. As such, a number of samples can be distributedacross a number of collecting containers, where the number of collectingcontainers is greater than the number of samples. Alternatively, thefirst substrate 110 can define a number of reservoir inlets greater thanthat of the number of collecting containers. As such, contents ofmultiple reservoirs can be combined prior to distribution across a setof collecting containers (e.g., in variations in which samples andprocessing materials are mixed within reservoirs prior to generation ofdroplets). As such, a reservoir number of the set of reservoirs can bedifferent from a collecting container number of the set of collectingcontainers, and the first substrate can include one or more fluidicpathways (or two or more fluidic pathways) from the set of reservoirs tothe set of membranes.

The first substrate 110 can include features configured to receive orposition the set of fasteners 140 (described in more detail below) forcoupling with other elements of the system 100, in order to enableretention of the elements of the system 100 in position relative to eachother. Such features maintain alignment and relative positioning betweensystem elements and provide proper sealing of elements (e.g., membranesto reservoir outlets). In the example shown in FIG. 2, the set offasteners 140 a can include a set of protrusions at the first substrate110 a, that complement tabs at the second substrate 160 a.

Alternatively, as shown in FIG. 3, the first substrate 110 c can includea set of flanges defining through-holes through the flanges with anorientation parallel to the broad surface of the first substrate 110 b,such that the set of fasteners including fastener 140 b pass parallel tothe broad surface through corresponding openings of a complementaryelement (e.g., second substrate 110 b, collecting container supportingbody, etc.). In this variation, separation of the first substrate 110from the complementary element would apply shear to the set offasteners.

Still alternatively, the first substrate 110 may not define any openingsor through holes, but enable fastening using another suitable mechanism.For instance, the first substrate 110 can include one or more of:protrusions (e.g., tabs, as shown in FIG. 4) that form a portion of asnap-fit mechanism between the first substrate 110 c and recesses of acomplementary element (e.g., second substrate 160 c, collectingcontainer supporting body, etc.); magnetic elements that enable magneticcoupling with a complementary element (e.g., second substrate 110,collecting container supporting body, etc.); adhesive elements thatcouple with a complementary element (e.g., second substrate 110,collecting container supporting body, etc.); and other suitable couplingmechanisms. The configurations shown in FIGS. 2, 3, and 4 providereliable coupling between elements of the system 100, without providingobstacles to accessing the reservoir(s) of the first substrate.

Additionally or alternatively, the first substrate 110 can includefeatures configured to receive or position the set of fasteners 140 forcoupling to other elements of the system 100, in order to enableretention of the elements of the system 100 in position relative to eachother. In one variation, the first substrate 110 can include a set ofthrough holes passing perpendicular to the broad surface of the firstsubstrate 110, in order to enable fastening between the first substrate110 and a complementary element (e.g., second substrate 110, collectingcontainer supporting body, etc.) with the membrane layer 120 andoptionally, the sealing body 130 positioned (e.g., sandwiched,compressed) between the first substrate 110 and the complementaryelement. In this variation, separation of the first substrate 110 fromthe complementary element would apply tension to the set of fasteners150.

2.1.2 Supporting Substrates—Second Substrate

In variations, the system 100 can additionally include a secondsubstrate 160, wherein the one or more membranes 120 and optionally, thesealing body 130 are retained in position between the first substrate110 and the second substrate 160 by the one or more fasteners 140described in more detail below. In variations, the second substrate 160can define openings (including second opening 60 shown in FIG. 1)corresponding to the set of reservoirs 114 and/or set of collectingcontainers 150, such that droplets formed upon transmission of fluidthrough the one or more membranes 120 pass through openings of thesecond substrate 160 and into the set of collecting containers 150. Theopenings of the second substrate 160 can further retain the collectingcontainers in position and/or in alignment with respective reservoiroutlets, such that generated droplets are transferred directly into thecollecting containers.

An example of the second substrate 160 is shown in FIG. 2, where the setof fasteners, including fastener 140 a, couples the first substrate 110a to the second substrate 160, with the membranes 120 a retained inposition between the first substrate no and the second substrate 160.The second substrate 160 a can be identical to or different from thefirst substrate 110 in material composition and/or properties describedabove. Furthermore, the second substrate 160 a can include or definechannels through which fluid passes into the collecting containers in adesired manner.

In the example shown in FIG. 2, the reservoir 114 a is partially formedby the first substrate 110 a and a reservoir channel 14 a, where thereservoir channel 14 a and the opening of the first substrate 110 acooperate to define a volume into which a material (e.g., sample fluid,process fluid, etc.), can be received for partitioning. As shown in FIG.2, the first substrate 110 a and the reservoir channel 14 a can besealed against each other (e.g., by a seal 130 a, by using compliantmaterials, such that biasing the first substrate 110 a against thereservoir channel 14 a forms a seal, etc.). Furthermore, as shown inFIG. 2, the membrane 120 a can be positioned at an outlet of thereservoir channel 14 a and seated within the second substrate 160 a, orpass through the second substrate into a respective collecting container150 a. In variations, the membrane 120 a can be bonded to the reservoiroutlet of the reservoir channel 14 a, or retained in position in anothermanner (e.g., by being seated between the reservoir channel 14 a and thesecond substrate 160 a).

While embodiments, variations, and examples of the first substrate 110described above include descriptions of a set of reservoirs, variationsof the first substrate 110 and/or second substrate 160 can alternativelydefine a single reservoir or pathway into a single collecting container.

2.2 System—Membrane

As shown in FIG. 1, the system 100 may include one or more membranes 120positioned adjacent to reservoir outlets of the set of reservoirs 114.The membrane layer 120 includes a distribution of holes 125, throughwhich fluid from the set of reservoirs 114 passes or is driven togenerate droplets for various applications. The one or more membranes120 can further function to provide a cost-efficient alternative tomicrofluidic devices for generation of droplets, where the one or moremembranes 120, along with other elements of the system 100 described,can generate droplets of a desired morphology (e.g., target droplet sizein relation to sample volume and target total number of partitions) in aconsistent (e.g., with high uniformity, with low merging, with lowpolydispersity) and reliable manner, and at an extremely rapid rate.

In relation to generation of droplets in a controlled manner, a membrane120 of the system 100 can include or be composed of one or moretrack-etched membranes (e.g., membrane sheets) with precisely definedhole sizes, membrane thicknesses and hole densities. For generation ofdroplets, fluid in the one or more reservoirs upstream of the membranelayer 120 is forced through the distribution of holes 125 by way ofapplied forces (e.g., centrifugation, pressurized gas, etc.). Thedroplets formed upon exit of the distribution of holes 125 are thentransmitted from the membrane 120 into one or more respective collectingcontainers described in more detail below, where the collectingcontainers contain a fluid (e.g., air, a fluid that is immiscible withthe droplet material, etc.). While the membrane 120 can be track-etchedto generate the distribution of holes in a precise manner (e.g., withrespect to size, shape, and density), the membrane 120 can alternativelybe generated without ion track techniques. For instance, the membrane120 can alternatively be generated using another method (e.g., laseretching, chemical etching, electroporation, etc.).

As shown in FIG. 1, the one or more membranes 120 are configured to beretained in position adjacent to the reservoir outlets 116 of the firstsubstrate 110. In variations, the membrane(s) 120 can thus be positionedwithin the reservoirs of the set of reservoirs 114 described above.Alternatively, the membrane layer 120 can be positioned outside of thereservoir outlets (e.g., at an external terminal portion of thereservoir 114, downstream of the reservoir outlets 116) of the firstsubstrate 110.

In variations, the membrane(s) 120 can include a continuum of materialpositioned across all of the one or more reservoir outlets of the set ofreservoirs, for instance, by sandwiching or otherwise retaining themembrane(s) 120 in position downstream of the reservoir outlets of thefirst substrate 110. Alternatively, the one or more membranes 120 caninclude separate bodies or regions of material positioned at terminalregions or within reservoirs of the first substrate 110. In variationswherein the one or more membranes 120 include separate bodies or regionsof material, the separate bodies/regions can each have differentcharacteristics (e.g., with respect to reservoirs carrying differenttypes of fluids, with respect to generation of different size droplets,etc.). As such, in some variations, each reservoir can have its ownassociated membrane region that is separate from adjacent membraneregions. In variations where the one or more membranes 120 are divided,individual membrane regions can be divided by a physical barrier (e.g.,a gap, a body of non-porous/non-absorbent material, etc.), or divided byanother suitable barrier.

The one or more membranes 120 can be coupled to other adjacent systemelements (e.g., the first substrate 110, reservoir outlets 116, optionalsealing body 130, the second substrate 160, etc.) by one or more of: anadhesive, a thermal bond, mechanical bond, chemical bond, laser welding,ultrasonic welding, a light-cured adhesive, a temperature-curedadhesive, a moisture-cured adhesive, injection-molding (e.g., as asingle piece using over-molding), and/or another suitable bondingmechanism.

FIG. 5 depicts an example method of bonding a membrane 120 d to areservoir outlet 16 d of a reservoir 116 d, at the perimeter of thereservoir outlet 116 d. As shown in FIG. 5, light-curable (e.g.,ultraviolet light-curable) adhesive 12 d is applied to the perimeter ofan exterior portion of the reservoir outlet 16 d. Then, material of themembrane (e.g., track-etched polycarbonate, track-etched polyethylene,etc.) is applied to the reservoir outlet 16 d at the perimeter, followedby application of a release film 17 d over the material of the membrane120 d. The light-curable adhesive 12 d is then exposed to light ofappropriate wavelength(s) (e.g., ultraviolet light), followed by removalof the release film 17 d and excess material of the membrane 120 d. Invariations, however, the membrane 120 can be coupled to the firstsubstrate 110 and/or the reservoir 116 in another suitable manner.

In variations, the one or more membranes 120 can be composed of apolymeric material (e.g., polycarbonate, polyester, polyimide). In afirst specific example, the one or more membranes 120 are polycarbonatetrack-etched (PCTE) membranes. In a second specific example, the one ormore membranes 120 are polyethylene track-etched membranes. However, theone or more membranes 120 can alternatively be composed of anothersuitable material processed in another suitable manner.

In variations, the distribution of holes 120 can be generated in bulkmembrane material with specified hole diameter(s), hole depth(s) (e.g.,in relation to membrane thickness), aspect ratio(s), hole density, andhole orientation, where, in combination with fluid parameters, thestructure of the membrane can achieve desired flow rate characteristics,with reduced or eliminated polydispersity and merging, and steadyformation of droplets (e.g., without jetting of fluid from holes of themembrane).

In variations, the hole diameter can range from 0.2 micrometers to 30micrometers, and in examples, the holes can have an average holediameter can be 0.02 micrometers, 0.04 micrometers, 0.06 micrometers,0.08 micrometers, 0.1 micrometers, 0.5 micrometers, 1 micrometers, 2micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers,7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20micrometers, 30 micrometers, any intermediate value, or greater than 30micrometers (e.g., with use of membrane having a thickness greater thanor otherwise contributing to a hole depth greater than 100 micrometers).

In variations, the hole depth can range from 1 micrometer to 200micrometers (e.g., in relation to thickness of the membrane layer) orgreater, and in examples the hole depth (e.g., as governed by membranethickness) can be 1 micrometers, 5 micrometers, 10 micrometers, 20micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100micrometers, 125 micrometers, 150 micrometers, 175 micrometers, 200micrometers, or any intermediate value.

In variations, the hole aspect ratio can range from 5:1 to 200:1, and inexamples, the hole aspect ratio can be 5:1, 10:1, 20:1, 30:1, 40:1,50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 125:1, 150:1, 175:1, 200:1, or anyintermediate value.

In variations, the hole density can range from 100 holes/cm² to 15000holes/cm² (e.g., in order to prevent droplets generated from neighboringholes from merging together as they exit the holes, in relation to spintime, speed, and/or pressurization, etc.). In examples, the hole densitycan be 100 holes/cm², 200 holes/cm², 300 holes/cm², 400 holes/cm², 500holes/cm², 600 holes/cm², 700 holes/cm², 800 holes/cm², 900 holes/cm²,1000 holes/cm², 2000 holes/cm², 3000 holes/cm², 4000 holes/cm², 5000holes/cm², 6000 holes/cm², 7000 holes/cm², 8000 holes/cm², 9000holes/cm², 10,000 holes/cm², 11,000 holes/cm², 12,000 holes/cm², 13,000holes/cm², 14,000 holes/cm², 15,000 holes/cm², or any intermediatevalue. In a specific example, the density of holes is less than 10,000holes/cm².

In variations, the hole-to-hole spacing can range from 5 micrometers to200 micrometers or greater, and in examples, the hole-to-hole spacing is5 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80micrometers, 90 micrometers, 100 micrometers, 125 micrometers, 150micrometers, 175 micrometers, 200 micrometers, or greater. In a specificexample, the hole-to-hole spacing is greater than 10 micrometers.

In variations, the membrane 120 can have a diameter or othercharacteristic dimension from 1 to 50 micrometers, and in examples, thediameter or other characteristic dimension can be 1 micrometer, 2micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers,7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20micrometers, 30 micrometers, 40 micrometers, 50 micrometers, or anyintermediate value.

In examples, the hole orientation can be substantially vertical (e.g.,during use in relation to a predominant gravitational force), otherwisealigned with a direction of applied force through the distribution ofholes, or at another suitable angle relative to a reference plane of themembrane layer 120.

In specific examples, the thickness of the membrane layer can be from23-125 micrometers, with a hole density of less than 10,000 holes/cm²and a hole diameter from 1 to 3 micrometers for generation ofwater-in-oil-in water (WOW) droplets approximately 14-30 micrometers indiameter under gravitational force of 16000 g without observation ofdroplet merging during formation. In the specific examples, dropletswere generated form a sample volume of 50 microliters, having a fluiddensity of 1255 kg/m³, a fluid viscosity of 0.007 Ns/m², and a surfacetension of 0.07 N/m. One specific example of the membrane 120 wascharacterized by a membrane thickness of 125 micrometers, a holediameter of 1.5 micrometers, a hole density of 5000/cm², an averagedroplet diameter of 30 micrometers, a polydispersity of ˜12.1 (CV, %),under a duration of centrifugation at 16,000 g for 10 minutes.

However, other fluid compositions and characteristics can be used, suchas those described in U.S. Pat. No. 11,162,136 granted on 2 Nov. 2021,which is herein incorporated in its entirety by this reference.

In relation to surface properties, the membrane layer 120 can be treatedor coated with a hydrophobic material in order to provide improvedconsistency of droplets (e.g., in relation to consistent droplet sizes,in relation to controlled droplet sizes, in relation to monodispersity,etc.). The hydrophobic material can additionally function to improveheat stability of droplets. In variations, the hydrophobic material caninclude one or more of: an oil, a polysiloxane (e.g., hydroxy-terminatedpolydimethylsiloxane), a fluorocarbon-coated silica with a polymerbinder; a perfluoroalkyl methacrylate copolymer with our without adistribution of substrates (e.g., nanoparticle substrates); apolystyrene material (e.g., manganese oxide polystyrene, zinc oxidepolystyrene), precipitated calcium carbonate, carbon nanotubes,fluorinated silanes, fluoropolymer coatings, silica-based coatings,nano-coatings, and/or other suitable hydrophobic or superhydrophobicmaterials. The material coating can be processed or otherwise selectedto produce desired characteristics in relation to contact anglecharacteristics (e.g., static contact angle, contact angle hysteresis),sliding angle characteristics, and/or other suitable characteristics.

In variations, the hydrophobic/superhydrophobic material can be appliedto the membrane layer 120 by one or more of: dip coating, spray coating,deposition (e.g., chemical vapor deposition), in-situ growth,polymerization, plasma coating, and/or another suitable coatingtechnique.

Additionally or alternatively, the membrane layer 120 can include or betreated with other components or coatings to provide desired functionsin relation to one or more of: electrostatic charge shielding (e.g., toprevent adsorption of proteins and other biomolecules, to improvedroplet stability), protection of the membrane layer (e.g., fromdegradation), and/or another suitable function.

Desired droplet sizes can be produced based upon a set of factorsassociated with the membrane layer 120 and applied forces. Parameters ofthe membrane layer 120 can be improved for generation of monodispersedroplets in relation to any one or more of Weber number; other factorsin relation to fluid inertia, surface tension, or other factors. In moredetail with respect to Weber number, the membrane layer 120 can beconfigured with a suitable characteristic hole dimension D (e.g., holedepth, hole diameter), intended fluid density ρ, governing fluidvelocity υ during droplet generation, and fluid surface tension 6, whereWeber number We=ρυ²D/σ, relating drag forces to cohesion forces inrelation to droplet generation from the membrane layer. In examples, Wesignificantly less than 1 produces periodic dripping for generation ofmonodisperse droplets, We˜equal to 1 produces chaotic dripping forgeneration of polydisperse droplets, and We greater than 1 producesjetting without droplet generation. In relation to membrane parametersdescribed above, increasing hole depth/thickness of the membrane 120from 50 micrometers to 125 micrometers decreases the We by approximately6.5 fold, and adjusting the hole diameter from 1 to 3 micrometerschanges We from 5.7e-6 to 1.395e-3.

Relatedly, parameters associated with the membrane layer 120, fluidcharacteristics, and applied force characteristics can be improved forgeneration of droplets having a desired size. In a variation for dropletformation with an air gap between the membrane layer 120 and acollection fluid within the collecting container, droplet radius R is afunction of hole radius r_(c), surface tension of the aqueous phaseγ_(aq), fluid density of the droplet fluid ρ_(w), and acceleration forceG (e.g., associated with applied centrifugation forces, associated withpressurization, etc.), where R˜[[r_(c)γ_(aq)]/[2ρ_(w)G]]^(1/3).

In a variation for droplet formation without an air gap between themembrane layer 120 and a collection fluid (e.g., an oil) within thecollecting container, droplet radius R is a function of hole radiusr_(c), surface tension of the interface) interfacial, fluid density ofthe droplet fluid ρ_(aq), fluid density of the collection fluid ρ_(oil),and acceleration force G (e.g., associated with applied centrifugationforces, associated with pressurization, etc.), whereR˜[[r_(c)γ_(interfacial)]/[2(ρ_(aq)−ρ_(oil))G]]^(1/3)

In specific examples, the resultant droplet size can be from 10micrometers to 80 micrometers; however, variations of the membrane layer120 and/or system 100, as well as applied force, fluid densities of thefluid to be dropletized and the receiving fluid, interfacial tension ofthe fluid to be dropletized, can be configured to generate droplets withany other suitable dimensions.

2.3 System—Seals

As shown in FIG. 1, the system 100 can optionally include one or moresealing bodies 130 positioned adjacent to the membrane layer 120 andincluding a set of openings 135 aligned with the set of reservoirs 114.The sealing body 130 functions to promote fluid transmission in adesired manner from the reservoir(s) of the first substrate 110 andthrough the membrane layer 120 to the collecting container(s), withoutleakage from the system 100 in an undesired manner. However, in somevariations, the system 100 can omit a sealing body, such as invariations where the membrane layer 120 is bonded to other systemelements directly, for instance, via light-curable bonding, heatbonding, or laser welding (e.g., an example of which is shown in FIGS.7A-7B and described below in relation to FIG. 5).

In variations, the sealing body 130 can include a first portion upstreamof the membrane layer 120. Additionally or alternatively, the sealingbody 130 can include a second portion downstream of the membrane layer.In such variations, the portion(s) of sealing body 130 can be compressedor otherwise retained against the membrane layer (e.g., byway of the setof fasteners 150, the first substrate 110, and/or the second substrate160). In these variations, the sealing body 130 portions can include orotherwise define openings aligned with and corresponding to the set ofreservoirs 114 or the set of collecting containers described in moredetail below. Additionally or alternatively, in a variation shown inFIG. 6, the sealing body 130 can additionally or alternatively include aportion (e.g., gasket or o-ring) positioned within a reservoir of thefirst substrate 110, in order to seal against a fluid delivery orpressurization device configured to drive fluid to or through themembrane layer 120 for droplet formation.

In variations, the sealing bodies 130 can include one or more of: anadhesive (e.g. optically-cured adhesive), heat-bonded seal, sealingelement (e.g., o-rings, gasket, etc.), sealing film or paste, or othersealing element.

2.4 System—Fastener(s) and Collecting Container(s)

As shown in FIG. 1, the system 100 can include one or more fasteners 140configured to retain the first substrate 110, the membrane layer 120,and the sealing body 130 in position relative to a set of collectingcontainers 150. The one or more fasteners 140 function to compress orotherwise retain elements of the system 100 in position properly, withrespect to forces applied to the system during droplet formation.

In variations, the fasteners 140 can include one or more of: screws,pins, plungers, protrusions (e.g., tabs), recesses (e.g., recessesconfigured to mate with protrusions), magnetic elements, adhesives,bonded couplers (e.g., thermally bonded couplers), and/or other suitablefasteners. Variations of fasteners are shown in FIGS. 2, 3, and 4 asdescribed above, wherein in the variation shown in FIG. 4, the fasteners140 include protrusions that form a portion of a snap-fit mechanism withrecesses of a complementary element (e.g., second substrate 110,collecting container supporting body, etc.).

As shown in FIGS. 1, 2, 6, and 7A-7C, the system 100 can be configuredto complement, mate with, or otherwise interface with one or morecollecting containers 150 for receiving generated droplets. FIG. 7Adepicts an example of the system 100, with a set of reservoirs,including reservoir 114 e defined within first substrate 110 e, where aset of membranes, including membrane 120 e, is bonded to reservoiroutlets of the set of reservoirs (e.g., using the process shown anddescribed in relation to FIG. 5). The system shown in FIG. 7A furtherincludes second substrate 160 e, which supports and aligns collectingcontainers, including collecting container 150 e, with the set ofreservoirs, such that the set of membranes is positioned within thecollecting containers during use of the system. During use, the firstsubstrate 110 e is thus assembled with the second substrate 160 e andcollecting containers, which is positioned within supporting body 170 efor application of a force to generate droplets.

In variations, the system 100 can be configured in a manner such thatthere is a gap (e.g., for air, for another fluid) between the system 100and the collecting container(s). In such variations, one of which isshown in FIG. 7B, the second substrate can be configured as a spacer 160f separating the membrane 120 f (at the respective reservoir outlet)from a base surface 50 f of the collecting container 150 f. As such,during droplet generation with one or more fluid layers within thecollecting container 150 f, the membrane 120 f can be spaced above afluid layer (FIG. 7B, top right), such that droplets generated from themembrane pass through air or other fluids prior to hitting the fluidlayer. However, the substrate 160 may not be configured as a spacer, asshown in FIG. 7B (bottom right), thereby positioning the reservoiroutlet 116 e directly within a fluid layer (e.g., non-aqueous phase, oilphase, for an aqueous sample fluid) for droplet generation. As such, thesystem 100 can be configured in a manner such that there is no gapbetween the system 100 and the collecting container(s) and generateddroplets are transmitted directly from the system into liquid layerswithin respective collecting containers.

In variations, the collecting container(s) can include any suitablenumber of containers with desired volumetric capacities. In examples(some of which are shown in FIGS. 7A-7C), the collecting container(s)150 can be configured with one or more of the following formats: 0.2 mltube format, strip tubes (e.g., 8× strip tube format), microtiter plateformat (e.g., 96-well plate format, 48-well plate format, 24-well plateformat, 12-well plate format, etc.), 1.5 ml tube format, conical tubeformat (e.g., 15 ml conical format, 50 ml conical format, etc.), oranother suitable format. The collecting container(s) can be disposableor reusable. In some variations, the collecting container(s) can besupported by a supporting body 170 configured to position the system 100properly with respect to force-applying apparatus. As shown in FIGS. 1and 2, the collecting containers can be supported by a supporting body170, 170 a, respectively, which can function as a swing bucket forpositioning of the system 100 within a centrifuge for force application,in order to generate droplets. However, the supporting body can beconfigured in another suitable manner.

The system 100 can, however, include other suitable elements forgeneration of droplets in a desired manner. Furthermore, the system 100can be configured to transition between various operation modes,including: a first operation mode wherein the first substrate is coupledwith the second substrate and encloses the collecting container, withthe reservoir outlet seated within the collecting container (an exampleof which is shown in FIG. 7A), a second operation mode wherein thereservoir contains a sample fluid (an embodiment of which is shown inFIG. 1), and a third operation mode wherein the membrane generates aplurality of droplets within the collecting container at a high rate(e.g., of at least 1 million droplets per minute, at other ratesdescribed) in response to a force applied to the sample fluid, and afourth operation mode wherein the plurality of droplets is stabilized inposition in a close-packed format within a region of the collectingcontainer. In relation to the operation modes described, structuralconfigurations of the system or contents of the system can producedroplets with a low degree of polydispersity (e.g., less than 15%coefficient of variation for polydispersity, less than 14% coefficientof variation for polydispersity, less than 13% coefficient of variationfor polydispersity, less than 12% coefficient of variation forpolydispersity, less than 11% coefficient of variation forpolydispersity, less than 10% coefficient of variation forpolydispersity, less than 9% coefficient of variation forpolydispersity, less than 8% coefficient of variation forpolydispersity, less than 7% coefficient of variation forpolydispersity, less than 6% coefficient of variation forpolydispersity, less than 5% coefficient of variation forpolydispersity, etc.) at an unprecedented rate, for digital analyses andother applications, as described above.

As such, generating the plurality of droplets can include driving theaqueous mixture through a distribution of holes of a membrane (e.g.,track-etched membrane) to stabilized positions toward a closed end ofthe collecting container, the plurality of droplets having significantlylow polydispersity, as described above.

Aspects of methods performed by embodiments, variations, and examples ofthe system 100 described in more detail below.

3. METHODS

As shown in FIG. 8, an embodiment of a method 200 for generation ofdroplets may include: generating a plurality of droplets within acollecting container at a high rate, each of the plurality of dropletsincluding an aqueous mixture for a digital analysis S210. Inembodiments, upon generation, the plurality of droplets is stabilized inposition in a close-packed format (e.g., three-dimensional close-packedformat, hexagonal close-packed format, rectangular close-packed format,etc.) within a continuous phase, within a region of the collectingcontainer S220.

Embodiments of the method 200 may function to generate a plurality ofdroplets at an extremely high and unprecedented rate in the context ofdigital analyses and other assays, where the droplets are stabilized inposition (e.g., in a close-packed format, in equilibrium stationarypositions) within a collecting container. Embodiments of the method 100may further function to reliably generate droplets in a consistent andcontrolled manner (e.g., as monodisperse and uniform droplets havinglittle-to-no polydispersity) for various applications, such as digitalamplification and analysis and other assays; capture of target materialat cellular, subcellular, and molecular scales; sample analysisbenefitting from droplet generation; and/or other suitable applications.Embodiments of the method may function to generate droplets usingdevices that are non-microfluidic, disposable or reusable, in acost-effective manner.

Embodiments, variations, and examples of the method 200 can beimplemented by or by way of embodiments, variations, and examples ofcomponents of the system 100 described in Section 2 above. However, themethod 200 can additionally or alternatively be configured to performother suitable methods.

3.1 Methods—Droplet Generation

In relation to generation of droplets at a high rate in Step S210,variations of the method 200 can produce droplets at a rate of at least200,000 droplets/minute, of at least 300,000 droplets/minute, of atleast 400, droplets/minute, of at least 500,000 droplets/minute, of atleast 600,000 droplets/minute, of at least 700,000 droplets/minute, ofat least 800,000 droplets/minute, of at least 900,000 droplets/minute,of at least 1 million droplets/minute, of at least 2 milliondroplets/minute, of at least 3 million droplets/minutes, or greater,using embodiments, variations, and examples of system elements describedabove. Droplets can be generated at the high rate, using embodiments,variations, and examples of the membrane(s) 120 described above, inrelation to hole density, hole-to-hole spacing, hole diameter, membranethickness, hole aspect ratio, membrane material, and/or othercharacteristics.

In relation to droplet generation in Step S210, an extremely high numberof droplets can be generated within a collecting container, wherein, invariations, greater than 2 million droplets, greater than 3 milliondroplets, greater than 4 million droplets, greater than 5 milliondroplets, greater than 6 million droplets, greater than 7 milliondroplets, greater than 8 million droplets, greater than 9 milliondroplets, greater than 10 million droplets, greater than greater than 15million droplets, greater than 20 million droplets, greater than 25million droplets, greater than 30 million droplets, greater than 40million droplets, greater than 50 million droplets, greater than 100million droplets, greater than 200 million droplets, greater than 300million droplets, or greater can be generated within the collectingcontainer.

In variations, the collecting container can have a volumetric capacityless than 50 microliters or from 50 through 300 microliters and greater.An example of a collecting container can include a PCR strip tube havinga volumetric capacity from 20 microliters to 50 microliters; however,other variations and examples of collecting containers are described inmore detail in Section 2 above. Droplets generated in Step S210 may havea characteristic dimension (e.g., from 1-50 micrometers, from 10-30micrometers, intermediate values within ranges described, etc.) that isrelevant for digital analyses, single cell capture, target detection,individual molecule partitioning, or other applications.

Generating the plurality of droplets in Step S210 can include driving asample fluid through a membrane comprising a distribution of holes, themembrane aligned with or coupled to a reservoir outlet of a reservoirfor the sample fluid. Driving the sample fluid can include applying acentrifugal force (e.g., by centrifugation) to drive the sample fluidthrough the holes of the membrane. In variations, the centrifugal forcecan be applied at 1,000 g, 2,000 g, 3,000 g, 4,000 g, 5,000 g, 6,000 g,7,000 g, 8,000 g, 9,000 g, 10,000 g, 11,000 g, 12,000 g, 13,000 g,14,000 g, 15,000 g, 16,000 g, 17,000 g, 18,000 g, 19,000 g, 20,000 g,30,000 g, any intermediate value, or greater than 30,000 g. Duration ofspinning can be 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, anyintermediate value, or greater than 50 minutes, where spin duration is afunction of the amount of sample fluid being dropletized according tomethods described.

In relation to generation of droplets of an emulsion (e.g., by drivingsample fluids through one or more immiscible layers of fluid to form amulti-phase emulsion), where resultant emulsions generated have viscousproperties, shear-thickening properties, and/or gel-like properties(e.g., as is present when generating emulsions where droplets are afirst phase surrounded by films of a second phase that is immisciblewith the first phase, and droplets are surrounded by a continuous thirdphase that is immiscible with the third phase), centrifugation canproduce emulsions having uneven top/superior surface (e.g., surfacesfurthest away from a base of the collecting container, along a forceaxis in a radial direction attributed to centrifugation), an example ofwhich is shown in FIG. 9A. Such uneven surfaces can increase difficultyof readout of signals from droplets near the top/superior surface (e.g.,emulsion surface furthest from the base of the collecting container),due to higher background, reduced clarity, and/or other factors.

As such, the method 200 can further include S214, which, as shown inFIG. 9B involves spinning the sample fluid, the membrane, and thecollecting container within a centrifuge in a first direction ofrotation, and reversing the direction of rotation, thereby adjusting asurface profile of an emulsion comprising the plurality of dropletswithin the collecting container. Adjusting the surface profile canimprove one or more of levelness, planarity, or other characteristics ofthe surface profile to improve readout ability. In relation to S214,spinning in the first direction and the second direction can beperformed at centrifugal forces in the ranges provided above or outsidedescribed ranges. Furthermore, spinning in the first direction can beperformed at a first rotational velocity, and spinning in the seconddirection can be performed at a second rotational velocity differentthan the first rotational velocity.

Additionally or alternatively, the method 200 can further include S216,which as shown in FIG. 9C, involves spinning the sample fluid, themembrane, and the collecting container within a centrifuge at a firstrotational velocity and at a second rotational velocity less than thefirst rotational velocity, thereby adjusting a surface profile of anemulsion comprising the plurality of droplets within the collectingcontainer. Adjusting the surface profile can improve one or more oflevelness, planarity, or other characteristics of the surface profile toimprove readout ability. In relation to S216, spinning at the firstrotational velocity and the second rotational velocity can be performedat centrifugal forces in the ranges provided above or outside describedranges. Furthermore, in relation to Steps S214 and S216, achieving thefirst rotational velocity and/or the second rotational velocity can beperformed with a ramp-up or acceleration rate, in order to improvesurface features of the emulsion.

In alternative variations, the applied force can be associated with anapplied pressure, magnetically applied, or otherwise physically appliedto drive sample fluid(s) through the membrane(s).

In relation to components of the sample fluid and/or fluid layers withinthe collecting container(s) for generation of an emulsion, the samplefluid and fluid layers within the collecting container can have one ormore of a certain density, viscosity, surface tension, aqueous nature,hydrophobicity, immiscibility characteristics, or other characteristics.Fluids implemented can have densities from 1 through 3000 kg/m³ andintermediate values, viscosities from 0.001 through 0.1 Ns/m², andsurface tensions of 0.01 through 1 N/m, depending upon application.Sample fluids and/or fluid layers can further include materialsdescribed in U.S. Pat. No. 11,162,136 granted on 2 Nov. 2021,incorporated by reference above. As such, droplets and/or resultingemulsions generated with said droplets can have a high degree andgreater than a threshold level of clarity, with or without refractiveindex matching. In variations, the threshold level of clarity of theemulsion is associated with a transmissivity greater than 50%transmissivity, greater than 60% transmissivity, greater than 70%transmissivity, greater than 80% transmissivity, greater than 90%transmissivity, greater than 95% transmissivity, greater than 99%transmissivity, etc., upon measuring clarity of the emulsion using atransmission detector.

In embodiments, upon generation, the plurality of droplets may bestabilized in position in a close-packed format (e.g., three-dimensionalclose-packed format, hexagonal close-packed format, rectangularclose-packed format, etc.) within a continuous phase, within a region ofthe collecting container S220. In relation to the membranes described,generating the plurality of droplets can include transmitting droplets(e.g., two dimensional arrays of droplets from the holes of themembrane(s)) toward a closed end of the collecting container, therebystabilizing the plurality of droplets in a three dimensionalclose-packed format toward the closed end of the collecting container.Alternatively, the plurality of droplets can be stabilized (e.g., withina continuous phase, within a matrix positioned within the collectingcontainer, within a mesh within the collecting container, etc.) towardthe closed end or a different region of the collecting container, in anon-close-packed format. For instance, non-close packed droplets ordroplets that can move relative to each other within the closedcollecting container can still be processed by optical interrogation asdescribed in more detail in Section 3 below (e.g., by fixing a positionof the closed collecting container relative to a scanning path of anoptical interrogation instrument). Additionally or alternatively, inrelation to close-packed or non-close-packed formats, droplets of anemulsion can be stabilized in position by curing (e.g., with light, withheat, with a pH shift, with other cross-linking, by way of an electricfield, by way of a magnetic field, etc.) the dispersed phase, continuousphase, or both of the emulsion. Still alternatively, droplets may not bestabilized in position or in a close-packed format (e.g., droplets canmove relative to each other within a container, such as for water-in-oilor oil-in-water emulsions, etc.).

In some variations, as shown in FIG. 8, the method 200 can furtherinclude: transmitting heat to and from the plurality of droplets, withinthe collecting container, during a heat transmission operation S230.Heat transmission can be associated with cold storage (e.g.,refrigeration, freezing, etc.), thermocycling (e.g., during anamplification process), incubation, lysis, enzyme activation, or anotherheat transmission operation. In variations, the temperature may varybetween 0° C. to 95° C. during the heat transmission operation, and inspecific examples, the temperature can transition between temperatureswithin the ranges described, with stability of droplets maintained. Inparticular, given the droplet generation techniques and materialsdescribed, individual droplets of the plurality of droplets remainunmerged with adjacent droplets in the close-packed format during theheat transmission operation.

In some variations, as shown in FIG. 8, the method 200 can furtherinclude: performing an optical interrogation operation with theplurality of droplets within the collecting container S240, where theoptical interrogation operation can include readout of signals (e.g.,light signals, fluorescent signals, colorimetric signals, etc.) fromdroplets of the plurality of droplets. In particular, readout can beperformed for cross sections of the plurality of droplets within thecollecting container, using techniques described in applicationsincorporated by reference.

In variations, readout of fluorescent signals (e.g., from labeledanalytes within droplets of the dispersed phase, from products ofanalytes within droplets of the dispersed phase, etc.) can be performedby one or more of a 3D scanning technique (e.g., light sheet imaging,confocal microscopy, etc.) and a planar imaging technique (e.g., to takeimages of a cross-section of the closed container). Additionally oralternatively, in a some applications, readout of colorimetric changesassociated with droplets of the dispersed phase can be performed by 3Dimaging techniques (e.g., 3D brightfield construction using light fieldimaging, etc.). Readout can be performed for each of a set of crosssections of the plurality of droplets/collecting container, acrossmultiple color channels (e.g., 2 color channels, three color channels,four color channels, five color channels, six color channels, sevencolor channels, etc.).

Readout can be performed for 10 cross-sections of the plurality ofdroplets, 20 cross-sections of the plurality of droplets, 30cross-sections of the plurality of droplets, 40 cross-sections of theplurality of droplets, 50 cross-sections of the plurality of droplets,60 cross-sections of the plurality of droplets, 70 cross-sections of theplurality of droplets, 80 cross-sections of the plurality of droplets,90 cross-sections of the plurality of droplets, 100 cross-sections ofthe plurality of droplets, 200 cross-sections of the plurality ofdroplets, 300 cross-sections of the plurality of droplets, 400cross-sections of the plurality of droplets, 500 cross-sections of theplurality of droplets, 600 cross-sections of the plurality of droplets,any intermediate value, or greater, within the closed collectingcontainer, for each of the set of color channels.

In specific examples, readout associated with digital analyses (e.g.,counting, quantification, etc.) for each channel can be performed withina duration of 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 30seconds, 20 seconds, 10 seconds, or less, depending upon one or more ofsignal-to-noise ratio, optical sensor sensitivity, excitation power(e.g., of a light source used to illuminate droplets and inducefluorescence), or other characteristics.

In other variations, readout of non-fluorescent signals from droplets ofthe dispersed phase can be performed. For instance, products resultingfrom reactions within individual droplets of the dispersed phase canproduce changes in one or more of refractive indices, light absorption,light scattering, light reflection, light transmission, or other lightinteraction characteristics that are different from empty or unreacteddroplets, for detection by various techniques (e.g., spectrophotometrictechniques, turbidimetric techniques, etc.).

As such, methods described enable digital analyses across a wide dynamicrange that is 10-100 times greater than that of existing technologies,depending upon application of use. In examples related to nucleic acidcounting, the methods disclosed herein can have a dynamic range from 1through 100 million, due to the extremely high number of uniformpartitions generated from which signals can be read, and due to theability to partition with low occupancy (e.g., less than 20% occupancy,less than 15% occupancy, less than 10% occupancy, less than 9%occupancy, less than 8% occupancy, less than 7% occupancy, less than 6%occupancy, less than 5% occupancy, etc.) of partitions by targets. Invariations, such low occupancy can enable characterization of targets ofinterest without requiring Poisson statistics-associated correctionfactors for partitioning error or other error.

In examples, generation of large numbers of droplets (as described)within a closed container can be performed within durations and at ratesdescribed (e.g., on the order of 1 million droplets/minute), and readoutof each channel for a digital analysis can be performed at a high rate(e.g., less than 1 minute per channel, across multiple color channels),thereby enabling readout for a digital analysis of millions ofpartitions on the order of minutes or hours (with time durationsdescribed as above).

As such, methods for droplet generation through readout of numbers ofdroplets described, for each of a set of channels for a digitalanalysis, can be performed within a duration of 1 minute, 2 minutes, 3minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, orany intermediate value. In examples, once droplet generation andamplification/tagging have been performed, readout of signals from eachchannel can be performed within 5 minutes, 4 minutes, 3 minutes, 2minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, or less,depending upon one or more of signal-to-noise ratio, optical sensorsensitivity, excitation power (e.g., of a light source used toilluminate droplets and induce fluorescence), or other characteristics.

As such, methods for droplet generation through readout of numbers ofdroplets can include: performing a digital analysis of target nucleicacid material from a sample within a duration (e.g., a durationdescribed), wherein performing the digital analysis includes: generatinga plurality of droplets (e.g., within a closed collecting container, theplurality of droplets comprising a number of droplets describedgenerated from a combination of the sample and materials for anamplification reaction, individually isolating the plurality of droplets(e.g., within a continuous phase of an emulsion), receiving heat (e.g.,through the closed collecting container), thereby amplifying said targetnucleic acid material, and transmitting signals, (e.g., from a set ofcross-sections of the emulsion comprising the plurality of dropletswithin the closed collecting container), for readout using a set ofchannels of a detection system (e.g., a detection system interactingwith the closed collecting container).

3.1.1 Method—Implementation

As shown in FIG. 10, an embodiment of a method 300 for generation ofdroplets includes: providing an assembly S310 including: a firstsubstrate defining one or more reservoirs, a membrane layer including adistribution of holes positioned downstream of the one or morereservoirs, one or more sealing bodies positioned adjacent to themembrane layer and including a set of openings aligned with the set ofreservoirs, and optionally one or more fasteners configured to retainthe assembly in position relative to one or more collecting containerscontaining a first fluid; optionally, receiving a second fluid into theone or more reservoirs S320, wherein the second fluid is intended foruse in droplet formation and is immiscible with the first fluid; andapplying force (e.g., centrifugation, pressurization, etc.) to contentsof the reservoirs/assembly S330, thereby driving the second fluid fromthe one or more reservoirs, through the membrane layer, and into the oneor more collecting containers.

Embodiments of the method 300 function to reliably generate monodispersedroplets for various applications (as described above, and at ratesdescribed above), such as digital amplification; capture of targetmaterial at cellular, subcellular, and molecular scales; sample analysisbenefitting from droplet generation; or other suitable applications.Embodiments of the method 300 can also function to generate monodispersedroplets using devices that are non-microfluidic, disposable, orreusable, in a cost-effective manner.

In specific applications, the method 300 can be used to generatedroplets with applications in one or more of: emulsion generation (e.g.,single emulsion generation, double emulsion generation), microparticlegeneration, liposome generation, hydrogel microparticle generation,nucleic acid amplification (e.g., by polymerase chain reaction (PCR)methods, by isothermal methods such as loop-mediated isothermalamplification (LAMP), by recombinase polymerase amplification (RPA), bymultiple displacement amplification (MDA), by helicase dependentamplification (HDA), by strand displacement amplification (SDA), bynicking enzyme amplification (NEAR), by transcription mediatedamplification (TMA), by digital helicase dependent amplification, byRNaseH mediated amplification, by whole genome amplification (WGA), byrolling circle amplification, etc.) on purified DNA, cDNA, RNA, ordirectly from lysate (e.g., blood lysate); fluorescent in situhybridization (FISH) with fluorescently tagged nucleic acids (e.g., PNA,LNA, DNA, RNA, etc.) or an indirect in situ hybridization approach usingDIG or biotin; by an in vitro transcription or translation assay (e.g.,whereby a colorimetric or fluorescent reporter is used for detection);droplet PCR applied to samples derived from single cells (e.g.,prokaryotes, eukaryotes), organelles, viral particles, and exosomes;droplet analysis of proteins (e.g., by proximity ligation assays, etc.);sequencing applications (e.g., single molecule sequencing applications);monitoring or detection of products (e.g., proteins, chemicals) releasedfrom single cells (e.g., interleukin released from immune cells);monitoring cell survival and/or division for single cells; monitoring ordetection of enzymatic reactions involving single cells; target materialcapture at cellular (e.g., mammalian cell, bacterial cell, pathogen,viral, etc.) and sub-cellular (e.g., organelle, molecular, etc.) scales;enumeration of heterogeneous cell populations in a sample; enumerationof individual cells or viral particles (e.g., by encapsulating cells indroplets with species-specific antibodies coupled with enzymes thatreact with substrate components in the droplet to produce signals,etc.); monitoring of viral infections of a single host cell; and othersuitable applications.

The method 300 can be implemented by an embodiment, variation, orexample of the system 100 described above, or can alternatively beimplemented by another suitable system.

4. COMPUTER SYSTEMS

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 11 shows a computer system1101 that is programmed or otherwise configured to, for example,generate a plurality of droplets within a collecting container at apredetermined rate or variation in polydispersity, transmit heat to andfrom the plurality of droplets within the collecting container, orperform an optical interrogation operation with the plurality ofdroplets within the collecting container.

The computer system 1101 can regulate various aspects of analysis,calculation, and generation of the present disclosure, such as, forexample, generating a plurality of droplets within a collectingcontainer at a predetermined rate or variation in polydispersity,transmitting heat to and from the plurality of droplets within thecollecting container, or performing an optical interrogation operationwith the plurality of droplets within the collecting container. Thecomputer system 1101 can be an electronic device of a user or a computersystem that is remotely located with respect to the electronic device.The electronic device can be a mobile electronic device.

The computer system 1101 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1105, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 101 also includes memory or memorylocation 1110 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1115 (e.g., hard disk), communicationinterface 1120 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1125, such as cache, othermemory, data storage and/or electronic display adapters. The memory1110, storage unit 1115, interface 1120 and peripheral devices 1125 arein communication with the CPU 1105 through a communication bus (solidlines), such as a motherboard. The storage unit 1115 can be a datastorage unit (or data repository) for storing data. The computer system1101 can be operatively coupled to a computer network (“network”) 1130with the aid of the communication interface 1120. The network 1130 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet.

In some embodiments, the network 1130 is a telecommunication and/or datanetwork. The network 1130 can include one or more computer servers,which can enable distributed computing, such as cloud computing. Forexample, one or more computer servers may enable cloud computing overthe network 1130 (“the cloud”) to perform various aspects of analysis,calculation, and generation of the present disclosure, such as, forexample, generating a plurality of droplets within a collectingcontainer at a predetermined rate or variation in polydispersity. Suchcloud computing may be provided by cloud computing platforms such as,for example, Amazon Web Services (AWS), Microsoft Azure, Google CloudPlatform, and IBM cloud. In some embodiments, the network 1130, with theaid of the computer system 1101, can implement a peer-to-peer network,which may enable devices coupled to the computer system 101 to behave asa client or a server.

The CPU 1105 may comprise one or more computer processors and/or one ormore graphics processing units (GPUs). The CPU 1105 can execute asequence of machine-readable instructions, which can be embodied in aprogram or software. The instructions may be stored in a memorylocation, such as the memory 1110. The instructions can be directed tothe CPU 1105, which can subsequently program or otherwise configure theCPU 1105 to implement methods of the present disclosure. Examples ofoperations performed by the CPU 1105 can include fetch, decode, execute,and writeback.

The CPU 1105 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1101 can be included in thecircuit. In some embodiments, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1115 can store files, such as drivers, libraries andsaved programs. The storage unit 1115 can store user data, e.g., userpreferences and user programs. In some embodiments, the computer system110 can include one or more additional data storage units that areexternal to the computer system 110, such as located on a remote serverthat is in communication with the computer system 110 through anintranet or the Internet.

The computer system 110 can communicate with one or more remote computersystems through the network 1130. For instance, the computer system 110can communicate with a remote computer system of a user. Examples ofremote computer systems include personal computers (e.g., portable PC),slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab),telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device,Blackberry®), or personal digital assistants. The user can access thecomputer system 101 via the network 1130.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 110, such as, for example, on the memory1110 or electronic storage unit 1115. The machine executable or machinereadable code can be provided in the form of software. During use, thecode can be executed by the processor 1105. In some embodiments, thecode can be retrieved from the storage unit 1115 and stored on thememory 1110 for ready access by the processor 1105. In some situations,the electronic storage unit 1115 can be precluded, andmachine-executable instructions are stored on memory 1110.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Embodiments of the systems and methods provided herein, such as thecomputer system 1101, can be embodied in programming. Various aspects ofthe technology may be thought of as “products” or “articles ofmanufacture” typically in the form of machine (or processor) executablecode and/or associated data that is carried on or embodied in a type ofmachine readable medium. Machine-executable code can be stored on anelectronic storage unit, such as memory (e.g., read-only memory,random-access memory, flash memory) or a hard disk. “Storage” type mediacan include any or all of the tangible memory of the computers,processors or the like, or associated modules thereof, such as varioussemiconductor memories, tape drives, or disk drives, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including a tangible storage medium, a carrier wavemedium or physical transmission medium. Non-volatile storage mediainclude, for example, optical or magnetic disks, such as any of thestorage devices in any computer(s) or the like, such as may be used toimplement the databases, etc. shown in the drawings. Volatile storagemedia include dynamic memory, such as main memory of such a computerplatform. Tangible transmission media include coaxial cables; copperwire and fiber optics, including the wires that comprise a bus within acomputer system. Carrier-wave transmission media may take the form ofelectric or electromagnetic signals, or acoustic or light waves such asthose generated during radio frequency (RF) and infrared (IR) datacommunications. Common forms of computer-readable media thereforeinclude for example: a floppy disk, a flexible disk, hard disk, magnetictape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any otheroptical medium, punch cards paper tape, any other physical storagemedium with patterns of holes, a RAM, a ROM, a PROM and EPROM, aFLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1101 can include or be in communication with anelectronic display 135 that comprises a user interface (UI) 1140 forproviding, for example, a visual display indicative of generating aplurality of droplets within a collecting container at a predeterminedrate or variation in polydispersity, transmitting heat to and from theplurality of droplets within the collecting container, or performing anoptical interrogation operation with the plurality of droplets withinthe collecting container. Examples of UIs include, without limitation, agraphical user interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1105. Thealgorithm can, for example, generate a plurality of droplets within acollecting container at a predetermined rate or variation inpolydispersity.

5. CONCLUSIONS

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of systems, methods and computer programproducts according to preferred embodiments, example configurations, andvariations thereof. In this regard, each block in the flowchart or blockdiagrams may represent a module, segment, or, if applicable, portion ofcode, which comprises one or more executable instructions forimplementing the specified logical function(s). It should also be notedthat, in some alternative implementations, the functions noted in theblock can occur out of the order noted in the FIGURES. For example, twoblocks shown in succession may, in fact, be executed substantiallyconcurrently, or the blocks may sometimes be executed in the reverseorder, depending upon the functionality involved. It will also be notedthat each block of the block diagrams or flowchart illustration, andcombinations of blocks in the block diagrams or flowchart illustration,can be implemented by special purpose hardware-based systems thatperform the specified functions or acts, or combinations of specialpurpose hardware and computer instructions.

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications may be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method comprising: performing a digitalanalysis of target nucleic acid material from a sample within a durationof 2.5 hours, wherein performing the digital analysis comprises:generating a plurality of droplets within a closed collecting container,the plurality of droplets comprising at least 20 million dropletsgenerated from a combination of the sample and materials for anamplification reaction, amplifying said target nucleic acid materialwith said materials for the amplification reaction, transmittingsignals, from a set of cross-sections of the emulsion comprising theplurality of droplets within the closed collecting container, forreadout using a set of channels of a detection system interacting withthe closed collecting container, and returning an analysis of saidsignals, the analysis supporting diagnosis of a genetic disorder.
 2. Themethod of claim 1, wherein the set of channels comprises four colorchannels for fluorescent detection of signals associated with saidtarget nucleic acid material.
 3. The method of claim 1, wherein thesample comprises a mixture of fetal genetic material and maternalgenetic material.
 4. A method comprising: performing a digital analysisof target nucleic acid material from a sample within a duration of 2.5hours, wherein performing the digital analysis comprises: generating aplurality of droplets within a closed collecting container, theplurality of droplets generated from a combination of the sample andmaterials for an amplification reaction, individually isolating theplurality of droplets within a continuous phase of an emulsion, andtransmitting signals, from a set of cross-sections of the emulsioncomprising the plurality of droplets within the closed collectingcontainer, for readout using a first channel and a second channel of adetection system interacting with the closed collecting container. 5.The method of claim 4, wherein generating the plurality of dropletscomprises generating the plurality of droplets at an average rate of atleast 1 million droplets per minute.
 6. The method of claim 4, whereinthe continuous phase comprises water.
 7. The method of claim 4, whereinthe emulsion is clear, and wherein clarity of the emulsion is associatedwith a transmissivity greater than 60% transmissivity of light withoutrefractive index matching of components of the emulsion.
 8. The methodof claim 4, wherein the set of cross-sections comprises at least 500cross-sections.
 9. The method of claim 4, wherein performing the digitalanalysis further comprises receiving heat, through the closed collectingcontainer, thereby amplifying said target nucleic acid material withsaid materials for the amplification reaction.
 10. The method of claim4, further comprising transmitting signals for readout using a thirdchannel, wherein readout for each of the first channel, the secondchannel, and the third channel is performed within a duration of 30seconds.
 11. The method of claim 10, further comprising transmittingsignals for readout using a fourth channel and a fifth channel.
 12. Themethod of claim 4, wherein performing the digital analysis furthercomprises returning results supporting prenatal diagnosis of a geneticdisorder.
 13. The method of claim 4, wherein performing the digitalanalysis further comprises returning results characterizing singlecells.
 14. A method comprising: performing a digital analysis of targetnucleic acid material from a sample, wherein performing the digitalanalysis comprises: generating a plurality of droplets within a closedcollecting container, the plurality of droplets comprising at least 10million droplets generated from a combination of the sample andmaterials for an amplification reaction, individually isolating theplurality of droplets within a continuous phase of an emulsion, andtransmitting signals, from a set of cross-sections of the emulsioncomprising the plurality of droplets within the closed collectingcontainer, for readout using a first channel and a second channel of adetection system interacting with the closed collecting container.
 15. Amethod comprising: performing a digital analysis of target nucleic acidmaterial from a sample within a duration of 2 hours, wherein performingthe digital analysis comprises: generating a plurality of dropletswithin a closed collecting container, the plurality of dropletsgenerated from a combination of the sample and materials for anamplification reaction, amplifying said target nucleic acid materialwith the amplification reaction, and transmitting signals, from a set ofcross-sections of the plurality of droplets within the closed collectingcontainer, for readout using a first color channel of a detection systeminteracting with the closed collecting container.
 16. The method ofclaim 15, wherein generating the plurality of droplets comprisesgenerating at least 20 million droplets within the closed collectingcontainer.
 17. The method of claim 15, wherein generating the pluralityof droplets comprises driving the combination through a distribution ofholes of a membrane under centrifugal force.
 18. The method of claim 17,wherein the membrane comprises a track-etched membrane and wherein thedistribution of holes comprises a hole-to-hole spacing greater than 30micrometers.
 19. The method of claim 15, further comprising transmittingsignals for readout using a second color channel, a third color channel,and a fourth color channel, wherein readout for each of the first colorchannel, the second color channel, the third color channel, and thefourth color channel is performed within a duration of 50 seconds. 20.The method of claim 15, wherein the set of cross-sections comprises atleast 400 cross-sections.